The reason for the high lethality of the 1918 influenza pandemic ("Spanish flu") remains one of the major unsolved questions in the field of human health. This devastating pandemic resulted in the deaths of many millions of people worldwide, causing unprecedented high mortality rates in healthy adults. Our lack of understanding of the virulence factors of the 1918 influenza virus that contributed to its high lethality makes it difficult to devise appropriate strategies of early diagnosis and prevention in the case of emergence of a 1918-like virus strain. In order to overcome this deficiency, we have put together a program project research plan with the goal of characterizing the genetic, structural, and molecular signatures of virulence of the 1918 influenza virus. Project 1, led by Dr. Taubenberger, is dedicated to obtaining the missing sequences of the 1918 virus and of pre- and post- 1918 influenza viruses from preserved human tissues. Project 2, which is led by Dr. Wilson, will determine the crystallographic structure of three 1918 viral proteins that are known to modulate virulence in several other influenza virus strains. Recently developed reverse genetics will be used to generate recombinant influenza viruses containing individual 1918 genes (or subsets of these genes). These viruses will be used by Drs. Basler, Palese and Garcia-Sastre, leaders of projects 3, 4 and 5, respectively, to investigate, at a molecular level, the sequence signatures of individual 1918 genes responsible for virulence, allowing for a complete functional analysis of the genome of the virus. These three projects will use a centralized Research Core, directed by Dr. Tumpey, to apply mouse and avian models of influenza virus infections to determine the role of the eight different 1918 genes in virulence. Experiments using recombinant viruses will be conducted in a USDA-approved Biological Safety Level 3 Agriculture (BSL3Ag) high containment facility. In addition, Project 6, led by Dr. Katze, will develop a non-human primate model of influenza virus pathogenicity (BSL3+) to overcome the limitations of other animal models of human influenza virus. Moreover, Dr. Katze will also apply micro-array-based computational technologies to determine the modulation of the host response during viral infection in vitro and in vivo. These studies will result in the discovery of patterns of host gene expression associated with severe influenza virus infection. By the multi-disciplinary approach used in this program project, we will be able to determine the main sequence/structure/function relationships responsible for the 1918 virus pathogenicity. This information will be critical to a better understanding of how to tackle highly lethal influenza viruses in the event of a new pandemic or of an intentional bioterrorism attack.